Project Summary Herpes simplex virus (HSV) infections remain a common, serious problem associated with significant morbidity. After primary infection, HSV establishes latency, which is not eliminated by current antiviral therapy. Latent virus is the source for viral reactivation and the recurrence of clinical disease. Despite much effort, a vaccine remains elusive. Therefore, there is a need for a novel therapeutic approach that would cure latent HSV infection. We have been developing a new curative strategy to latent HSV infection, in which an HSV-targeted endonuclease induces mutagenesis of essential HSV genes, disabling viral genomes and rendering the virus incapable of replication or reactivation from latency. Our hypothesis is that recent advances in the field of gene-editing technologies and in vivo gene delivery offer the opportunity to improve our current anti-viral approach and reach therapeutic efficacy. Here, we propose to evaluate Staphylococcus aureus (Sa)CRIPSR/Cas9 and homing endonucleases (HEs) for their ability to disable HSV in latently infected neurons by targeted viral genome disruption, thus eliminating the source of viral pathogenesis in an animal model of HSV infection. The goal of this project is to maximize the efficacy and safety of our approach to eliminate latent HSV infection in vivo, using a murine model of HSV latent infection. Our results will also be applicable in efforts to cure varicella zoster virus, another alphaherpesvirus that like HSV establishes latency in sensory neurons. Furthermore, the data generated will be highly relevant to the development of cures for other chronic or latent viral infections such as hepatitis B virus, HIV, or human papillomavirus. In SA1: Address the remaining barriers to effective in vivo HSV gene editing, we will compare 1) new AAV serotypes to our current AAV serotypes for transgene delivery after administration via different routes, and 2) the gene editing abilities of CRISPR/Cas9 vs. HEs. In SA2: Optimize the efficacy of in vivo gene editing and determine the impact on viral pathogenesis, we will evaluate whether the simultaneous targeting of two HSV sites provides superior efficacy over targeting a single site, and the efficacy of gene editing necessary to impact viral pathogenesis. In SA3: Evaluate the safety of in vivo gene editing in our mouse model, we will use our mouse model of latent HSV infection to evaluate tolerability, safety, and genotoxicity of nuclease exposure. This project is expected to demonstrate the feasibility of our therapeutic approach directed towards the elimination of HSV pathogenesis in vivo, and to provide critical information for the development of a larger scale animal study necessary to bring this new therapeutic approach to the clinic.